MAPPING EPITOPES OF HUMAN FC-GAMMA-RII (CDW32) WITH MONOCLONAL-ANTIBODIES AND RECOMBINANT RECEPTORS

FcgammaRII is a low affinity FcR for IgG with two Ig-like extracellular domains (D1gamma and D2gamma), a transmembrane domain, and a cytoplasmic domain. The production, characterization, and epitope analysis of four anti-human FcgammaII mAb (8.2, 8.7, 8.26, and 7.30) are detailed, and the mAb are compared with two defined CDw32 mAb, IV.3 and CIKM5. Reactivity of all mAb with FcgammaRII was demonstrated by (a) specific binding to FcgammaRII+ L cells (produced after transfection of L cells with human FcgammaRIIa cDNA, HFc3.0), by using flow cytometry, (b) inhibition of the binding of SRBC sensitized with rabbit antibody (EA) to FcgammaRII+ L cells, and (c) immunoprecipitation and SDS-PAGE, which detected a 42-kDa protein on K562 and U937 cells and a single 45-kDa protein on FcgammaRII+ L cells. The mAb were able to detect different forms of FcgammaRII, by flow cytometry, on Daudi cells (8.7 and 7.30) and U937 cells (8.2, IV.3, and CIKM5); 8.26 stained Daudi cells with intermediate fluorescence and U937 cells with the highest fluorescence, relative to the remaining mAb. Binding to transiently expressed isoforms of FcgammaRII (a and b1) and four allelic variants of FcgammaRIIa in COS-7 cells did not distinguish the mAb epitopes. Further mapping of the mAb epitopes was determined by (a) EA inhibition assays, (b) mAb blocking studies, and (c) the binding of the mAb to segments of human FcgammaRIIa by using genetically engineered chimeric receptors. Chimeric receptors expressing either D1gamma linked to domain 2 of FcepsilonRI or domain 1 of FcepsilonRI linked to D2gamma were produced by exchanging homologous, but antigenically different, regions of FcgammaRIIa and the high affinity receptor for IgE. Four clusters of mAb were identified, each mapping to discrete epitopes of FcgammaRII. Cluster I (mAb 8.2 and CIKM5) defines a combinatorial epitope with determinants in D1gamma and D2gamma distant from the IgG Fc binding site, inasmuch as F(ab')2 fragments of 8.2 and CIKM5 do not inhibit the binding of EA to FcgammaRII. The epitopes of clusters 2 (mAb 8.26), 3 (mAb IV.3), and 4 (mAb 8.7 and 7.30) are located entirely in D2gamma and all involve the IgG Fc binding region, because F(ab')/F(ab')2 fragments of the mAb inhibit EA binding to FcgammaRII. Thus, all mAb that inhibit the binding of EA map totally to D2gamma; it is likely the IgG Fc binding region is also contained in D2gamma.